Sediment is an important factor affecting the growth of aquatic plants. The effects of sediment substrate application on aquatic plants and rhizosphere microorganisms are poorly understood. In this study, we investigated the effects of four substrates (maifanite, vermiculite, ceramsite, and volcanic rock gravels) on the growth and rhizosphere microorganisms of Vallisneria natans (Lour.) Hara (V. natans) by Illumina high-throughput sequencing technology. The results showed that the maximum increase in plant height (20.5 cm) was observed in vermiculite group during the whole experiment period (day 1-40). The root activity of V. natans in the four treatment groups (34.78~85.28 U g-1) was higher than that in CK (28.48 U g-1). The maximum mean total chlorophyll content in maifanite group was 2.40 mg g-1, followed by vermiculite group (1.1 mg g-1). Catalase activity in leaves in all the treatment groups except maifanite group was significantly different during different periods (P<0.05). The maximum mean superoxide dismutase activity in maifanite group was 273.78 U g-1 min. All four treatment groups exhibited faster reduction in malondialdehyde content in leaves than CK group, indicating that substrate addition could rapidly enhance plant stress adaptation. Redundancy analysis showed that root vitality was positively correlated with biomass, plant height, and root length, and that different substrates had different effects on the growth of V. natans. Dominant bacterial phyla in rhizosphere sediments in each group were roughly the same, and the top two bacterial phyla were Proteobacteria and Chloroflexi. Relative abundance of Desulfobacterota and Nitrospirota were higher in four treatment groups than in CK group. Our results showed that substrate addition, especially maifanite, into lake sediments could promote the growth of submerged plants and improve the rhizosphere microbial community structure. Our findings provide theoretical basis for lake sediment improvement and aquatic vegetation restoration.